REPORTS 21.5
20.5 21
19.5 20
18.5 19
18 0 5 10 15 20 Wells well A1 (active wells are illustrated in Supplementary Figure S1). Melting curve analysis was performed to confirm amplification of a single product (data not shown).
wells, meaning that the sample is only 10 µm from the heat source (Figure 1A). To enable thermal uniformity across all of the samples to within ± 0.3°C during holds at a static temperature or within ± 0.8°C during fast ramping (10°C/s), the xxpress uses an array of infrared sensors to determine the temperature of the test samples, and a control algorithm adjusts the heating patterns at a rate of 100 times per second. The xxplates to be analyzed using the xxpress system were prepared as follows: Plates were sealed using xxpress compatible polarized sealing strips heated at 170°C for 1.5 s using the heat sealer provided (Figure 1B). They were then centrifuged at 1000 rpm for 1 min in dedicated holders in
Table 2. PCR cycling conditions. xxpress
Initial denaturation Denaturation
Annealing extension* Cooling
20 1
10 10
10 (4.8† 10 (4.8† 10 (4.8† 10 (4.8†
) ) ) )
95 95 60 50
LightCycler 480
Temp (°C) Hold (s) Ramp rate (°C/s) Temp (°C) Hold (s) Ramp rate (°C/s) 95 95 60 50
20 1
10 10
*Fluorescence was measured following each annealing and extension step. †
Vol. 58 | No. 5 | 2015
4.8 4.8 2.5 2.5
Cycling conditions for PCR. Ramp rates are shown for fast PCR and conventional PCR in parentheses. Ramp rate used to assess consistency across the plate by amplification of 18S rDNA.
246
the centrifuge provided before loading into the xxpress cycling unit
(Figure
1B). The LightCycler 480 system with a 384-well block installed has a thermal uniformity of ± 0.4°C within 60 s of target attainment (72°C). The PCR plates (Roche) to be analyzed were prepared following the manufac- turer’s instructions (LightCycler 480 user manual). In brief, plates were sealed using the manufacturer’s sealing strips, ensuring all wells were securely covered, followed by centrifugation at 1200 rpm for 2 min. Plates were then loaded into the LightCycler.
PCR conditions and data analysis All PCR reactions were performed for 40 cycles with SYBR Fast qPCR
Mastermix (Kapa Biosystems, Woburn, MA) (final concentration 1×; 1 µL DNA or cDNA template, 5 µM forward and reverse primers) in a final volume of 5 µL. The cycling conditions used for amplification of 18S rDNA are shown in Table 2 and employed the same ramp rate (4.8°C/s) on both machines. 18S rDNA amplification used 1 µL of genomic DNA as template (2.5 ng/µl), either undiluted or serially diluted in PCR grade water. The same condi- tions were used for detection of miRNAs but with a reduced annealing/ extension temperature of 55°C and with ramp rates increased to 10°C/s on the xxpress. Raw fluorescence data were analyzed by baseline normal- ization, and Cq
values were called in
accordance with the data analysis software provided on each instrument (threshold set using the second deriv- ative maximum algorithm on the Light- Cycler and manually on the xxpress).
RNA templates An RNA oligonucleotide with the sequence of miR-21 and a poly(A) tail (miRNA mimic) (Table 1) was reverse transcribed using 5 pmol of oligodT- RACE pr imer. The template was
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Figure 2. Variability in Cq values across plates on both systems. A single 18S rDNA PCR mastermix was distributed across 48 wells of both a Lightcycler 384-well plate and an xxpress 96-well xxplate (5 µL reaction/well). PCR was performed with the same ramp rate on each thermal cycler (4.8°C/s). Cq
values are presented by row from 25 30 35 40 45 50
xxpress Lightcycler
Cq
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